| Nitric oxide (NO) is a ubiquitous signaling molecule involved in plant development and defense processes, including pests and diseases, high temperature, drought, water stress, salt stress and metal toxicity. Moreover, NO acts as a signal in aluminum (Al) tolerance by triggering hypersensitive responses. Whereas the data on the regulating role of NO on heavy metal toxicity in plants is accumulating, but the physiological role of NO in alleviating Al toxicity is still poorly understood. In this study, we investigated the endogenous NO sources and components with two soybean cultivars differing in Al tolerance to identify the defense role of NO in Al tolerance. Furthermore, the effects of NO donor (SNP) and NO scavenger (cPTIO) on Al-induced oxidative stress and cell wall rigidity were also investigated. Specific findings are performed as follows:(1) To evaluate the role of NO in alleviating Al toxicity, we inveatigated the responses of endogenous NO level and reactive oxygen species (ROS) content in root tips of two soybean cultivars with significant differences in Al tolerance. The results showed that50μmol·L-1Al remarkably induced the NO and ROS production in soybean root tips within48h, and endogenous NO level was higher in ZheQiu No.2than in ZheChun No.3, O2-and H2O2content in ZheQiu No.2was lower by15.54%and9.04%than in ZheChun No.3. Superoxid dismutase (SOD)、peroxidase (POD)、 glutathione peroxidase (GPX) and catalase (CAT) activity in ZheQiu No.2was increased by95.31,122.98,11.19and191.56%compared to ZheChun No.3. The inhibition rate of root elongation and Al accumulation in root apex of ZheQiu No.2was lower than ZheChun No.3. These results suggested that Al induced the accumulation of NO and ROS in soybean, the production of NO and scavenging ability of ROS in Al-tolerant cultivar was greater than in Al-sensitive cultivar. Al-tolerant cultivar ZheQiu No.2accumulated more NO and less ROS, namely they suffered from less oxidative stress. Above results suggested that, NO played a role in Al tolerance by alleviating Al-induced oxidative stress.(2) To elucidate the endogenous NO sources involved in Al tolerance in soybean root tips, a NO synthase (NOS) inhibitor Nu-nitro-L-arginine (L-NNA) and a nitrate reductase (NR) inhibitor (Na2WO4) on Al toxicity were investigated with ZheChun No.3cultivar. There were no significant difference between L-NNA pretreatment and Al treatment alone with the relative root elongation, Al accumulation in root apex and NO content. While, compared with the Al treatment alone, the relative root elongation and NO content pretreated by Na2WO4decreased, and Al accumulation in root apex increased. Furthermore, NR activity significantly decreased by64.79%in Na2WO4pretreatment compared with the Al treatment alone, and NR activity in L-NNA pretreatment decreased by11.68%. While the NOS activity under Al stress were undetectable. All of these results indicated that NR-dependent NO production plays an important role in providing protection against Al toxicity.(3) To investigate the effect and regulative mechanisms of NO on Al-induced oxidative stress, NO donor (SNP) and NO scavenger (cPTIO) were pretreated to regulate NO level in ZheChun No.3cultivar. The inhibition rate of root elongation, the degree of membrane injury and Al accumulation in root apex were decreased by NO donor pretreated, indicating NO improved Al tolerance. Exogenous NO also increased the antioxidant enzyme (SOD, POD, CAT and GPX) activity, decreased the O2-accumulation. While H2O2accumulation in root apex enhanced. These NO effects were reversed by NO scavenger (cPTIO). The results suggested that NO improved Al tolerance in soybean by maintaining protective enzyme activity and reducing oxidative damage, and H2O2might acted as signaling molecule.(4) To identify NO, H2O2and their combination within plant signaling probably participated in the plant and cellular resistant responses of soybean plants under Al toxicity conditions, seedlings were pretreated with exogenous NO and H2O2. Al treatment significantly increased endogenous NO and H2O2in soybean root tips. And by supply exogenous NO donor and H2O2could effectively alleviated the Al-induced inhibition of root elongation, Al accumulation in root tips and Al-induced death of RBCs. And NO scavenger (cPTIO) and H2O2scavenging enzymes (CAT) could clear and even reversed the mitigation effects of NO and H2O2. These results showed that NO and H2O2had a positive role in inducing the resistance of RBCs and soybean root tips to Al. Further tests founded that, NO-induced accumulation of H2O2in soybean root tips; H2O2had no significant effect on the accumulation of NO in soybean root tips. According to the above results suggested that NO induced the resistance of RBCs and soybean root tips to Al by regulating the production of H2O2. And, H2O2may locate in the downstream of signaling is regulated by NO, it resist Al toxicity damage by activating antioxidant defense responses or programmed cell death signaling pathways.(5) To investigate the mechanisms of NO medicated the cell wall expansion in Al tolerance of soybean; NO donor (SNP) and NO scavenger (cPTIO) were pretreated with ZheChun No.3cultivar. Exogenous NO decreased the cell wall peroxides (cPOD), laccase and oxalate oxidase (OXOs) activites, reduced H2O2content and lignin accumulation. And these effects were reversed by NO scavenger cPTIO. Exogenous NO pretreatment also significantly reduced the Al accumulation in root apex and root cell wall, increased polygalacturonase (PG) activity, and decreased the cell wall component pectin, hemicellulose2and pectin methyl esterifying enzyme (PME) activity. And these effects were reversed by NO scavenger cPTIO. These results suggested that NO decreased Al-induced lignin deposition then to extense cell wall, and NO reduce the Al accumulation in cell wall by maintain a lower cell wall component (pectin, hemicellulose2) and higher PME activity, then to avoid the change of the hardness, rigid structure and properties of cell walls caused by Al binding. |
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